Phytochemical and In Silico ADME/Tox Analysis of Eruca sativa Extract with Antioxidant, Antibacterial and Anticancer Potential against Caco-2 and HCT-116 Colorectal Carcinoma Cell Lines

Eruca sativa Mill. (E. sativa) leaves recently grabbed the attention of scientific communities around the world due to its potent bioactivity. Therefore, the present study investigates the metabolite profiling of the ethanolic crude extract of E. sativa leaves using high resolution-liquid chromatography-mass spectrometry (HR-LC/MS), including antibacterial, antioxidant and anticancer potential against human colorectal carcinoma cell lines. In addition, computer-aided analysis was performed for determining the pharmacokinetic properties and toxicity prediction of the identified compounds. Our results show that E. sativa contains several bioactive compounds, such as vitamins, fatty acids, alkaloids, flavonoids, terpenoids and phenols. Furthermore, the antibacterial assay of E. sativa extract showed inhibitory effects of the tested pathogenic bacterial strains. Moreover, the antioxidant activity of 2,2-diphenyl-1-picrylhydrazyl (DPPH) and hydrogen peroxide (H2O2) were found to be IC50 = 66.16 μg/mL and 76.05 μg/mL, respectively. E. sativa also showed promising anticancer activity against both the colorectal cancer cells HCT-116 (IC50 = 64.91 μg/mL) and Caco-2 (IC50 = 83.98 μg/mL) in a dose/time dependent manner. The phytoconstituents identified showed promising pharmacokinetics properties, representing a valuable source for drug or nutraceutical development. These investigations will lead to the further exploration as well as development of E. sativa-based nutraceutical products.


Introduction
Eruca sativa Mill. (Eruca vesicaria) is a very popular species of the Brassicaceae family, which is commonly known as arugula, jarjeer or rocket leaves [1,2]. It is also popularly used in salad preparations. E. sativa leaves are considered as a valuable source of nutrition due to the presence of several important nutrients, such as dietary fiber, oligosaccharides, amino
The presence of different types of phytochemicals, such as flavonoids, phenolics, tannins, saponins and essential oils, are also reported in the literature [1,19,24,25]. The chemodiversity of E. sativa essential oils possess 4-methylthiobutyl isothiocyanate, 5-methylthiopentanonitrile, as well as a large amount of sulfur-and nitrogen-containing compounds. Erucic acid is a major component of the plant, along with glucosinolatemethyl sulphinyl butyl isothiocyanate [22]. Furthermore, Jirovetz et al. (2002) analyzed the aromatic compounds of the fresh leaves E. sativa using gas chromatography. They reported the presence of various components, such as iso-thiocyanates, derivatives of butane, octane, nonane, 4-methylthiobutyl isothiocyanate, cis-3-hexen-1-ol,5-methylthiopentylisothiocyanate, cis-3-hexenyl 2-methylbutanoate, and 5-methyl thiopentone nitrile [6]. Zhang and Tang (2007) found that E. sativa leaves contain volatile components and the major constituents identified were 4-methylthiobutyl isothiocyanate, 5-methylthio-pentanonitrile and abundant amounts of sulfur-and nitrogen-possessing compounds. They also reported that isothiocyanates (isothiocyanate and erucin) had cytoprotective effects [26]. Nazif et al. (2010) investigated a 70% alcoholic extract of E. sativa seeds, which led to the isolation of three flavonoids, quercetin, rhamnetin and kaempferol-3-O-galactoside, in addition to two glucosinolates; 4-(methylthio)butyl-glucosinolate (glucoerucin) and 3-methylsulfinylpropylglucosinolate (glucoiberin) [7]. Michael [19].     Arora et al. (2014) revealed that glucosinolates and their hydrolytic products form a significant class of plant secondary metabolites involved in numerous plant defenselinked mechanisms. They exploit the volatile nature of the glucosinolates and develop a method that not only enhance the yield of glucosinolate hydrolytic products, but also reduce the amount of undesired compounds. Among all the tested protocols, the hydrodistillation method using the Clevenger apparatus was considered as the best procedure, which was evident from an enhanced yield as well as an increased number of hydrolytic products when compared to the other methods as observed by gas chromatographymass spectrometry (GC-MS) [27]. Hussein et al. (2014) revealed that naturally occurring polyphenolic compounds, such as flavonoids, were abundantly present in E. sativa, which has a wide range of therapeutic properties (for instance, antimicrobial, anti-allergic, antiinflammatory, antioxidant and anticancer). Among different classes of flavonoids, the flavonol class was found to be the most important and widely spread flavonoid, in which quercetin, kampferol, rutin and myricetin were found to be the most potent [8]. Cavaiuolo et al. (2014) described E. sativa as an important leafy vegetable crop and a good source of antioxidants and anticancer molecules, such as glucosinolates and other sulfur compounds. E. sativa is also a hyper-accumulator of nitrates, which have been considered for a long time as the main factors that cause gastro-intestinal cancer. Moreover, recent studies carried out on E. sativa determined that the consumption of leafy vegetable would reduce the risk of contracting cancer and other cardiovascular diseases [28].

Antibacterial Activity of E. sativa Crude Extract
The antibacterial activity of E. sativa crude extract was evaluated via the agar welldiffusion assay method against two strains of Gram-positive pathogenic bacteria (B. subtilis and S. aureus) and two strains of Gram-negative pathogenic bacteria (E. coli and P. aeruginosa). Our results show that the crude extract of E. sativa exhibits a substantial inhibition of all the tested pathogens. The results of the antibacterial activity as well as the depiction of the crude extract preparation are presented in Figure 3. The obtained results reveal that E. sativa crude extract is potentially effective in controlling bacterial growth, and evaluated the antioxidant activity of the methanolic-ethanolic extracts of E. sativa leaves. The qualitative and quantitative estimations of quercetin, kaempferol, rutin and myricetin were reported by thin layer chromatography (TLC) and high-performance liquid chromatography (HPLC), revealing that kaempferol had the highest concentration followed by myricetin and rutin, while quercetin had the lowest. It was reported that the antioxidant activity of the E. sativa extract was more reactive at a concentration of 20 µg/mL, and less active at a concentration of 2.5 µg/mL (IC 50 33 and 47 µg/mL, respectively). Moreover, E. sativa has come into the limelight due to its high and significant amount of phytochemicals, which is still under investigation [3].

Antibacterial Activity of E. sativa Crude Extract
The antibacterial activity of E. sativa crude extract was evaluated via the agar welldiffusion assay method against two strains of Gram-positive pathogenic bacteria (B. subtilis and S. aureus) and two strains of Gram-negative pathogenic bacteria (E. coli and P. aeruginosa).
Our results show that the crude extract of E. sativa exhibits a substantial inhibition of all the tested pathogens. The results of the antibacterial activity as well as the depiction of the crude extract preparation are presented in Figure 3. The obtained results reveal that E. sativa crude extract is potentially effective in controlling bacterial growth, and E. sativa crude extract shows a maximum zone of inhibition against S. aureus, followed by B. subtilis, E. coli and P. aeruginosa.  Currently, the antibiotic resistance of pathogenic bacteria is a major concern around the globe, where multidrug-resistant bacteria are continuously emerging and spreading causing a great challenge to the healthcare system. [9]. Therefore, there is always a great demand for the search for an effective novel antimicrobial agent, which can fight against multidrug-resistant bacteria. Plants, especially medicinal/aromatic, are considered as a potential source of antimicrobial agents, as they consist of a high number of diverse phytochemicals [1]. In the present study, the antibacterial activity of E. sativa crude extract was evaluated using the agar well-diffusion method against different pathogenic strains of bacteria. Among them, the E. sativa crude extract exhibited the considerable inhibition of all the tested pathogens. Currently, the antibiotic resistance of pathogenic bacteria is a major concern around the globe, where multidrug-resistant bacteria are continuously emerging and spreading causing a great challenge to the healthcare system [9]. Therefore, there is always a great demand for the search for an effective novel antimicrobial agent, which can fight against multidrug-resistant bacteria. Plants, especially medicinal/aromatic, are considered as a potential source of antimicrobial agents, as they consist of a high number of diverse phytochemicals [1]. In the present study, the antibacterial activity of E. sativa crude extract was evaluated using the agar well-diffusion method against different pathogenic strains of bacteria. Among them, the E. sativa crude extract exhibited the considerable inhibition of all the tested pathogens.
Using the agar cup diffusion method, Kauba et al. (2015) reported an inhibition zone (IZ) similar to the one obtained in our results with E. sativa ethanolic extract against S. typhimurium (IZ = 16.7 mm), B. subtilis (IZ = 16.6 mm), E. coli (IZ = 16.0 mm) and B. thuringiensis (IZ = 15.6 mm) [14]. Additionally, Khoobchandani et al. (2010) reported the antimicrobial activity of crude extracts of different parts of E. sativa against two Grampositive and three Gram-negative bacteria [29]. Among them, a higher activity was reported for the seed oil against Gram-positive bacteria compared to Gram-negative bacteria. Additionally, Qaddoumi and El-banna (2019) reported the antagonistic activity of the aqueous extract of E. sativa towards E. coli (IZ = 19.0 mm) and S. aureus (IZ = 12.0 mm) [30]. In the same study, the antimicrobial activity of the crude extract of ethyl acetate presented no antimicrobial activity towards the tested pathogens. In another study, Rizwan et al.
(2016) reported the antimicrobial activities of ethanol, methanol, ethyl acetate, acetone and chloroform extracts from E. sativa against different Gram-positive and Gram-negative bacteria [31]. Among them, a higher inhibition activity was found in the ethyl acetate and chloroform extracts against S. aureus (IZ = 25.66 mm, 23.16 mm), respectively, followed by methanol and ethanol (IZ = 16 mm, 14.33 mm).

The Antioxidant Activity of E. sativa Crude Extract
Free radicals, such as reactive oxygen species (ROS) and reactive nitrogen species (RNS), are linked with oxygen, which possess a strong reaction activity with other molecules apart from oxygen. Frequently, such free radicals are raised as a by-product of different metabolic activity inside the cell and ionize radiation in the form of the hydroxyl radical (HO•); superoxide ion, O 2 −; singlet oxygen (O 2 ); hydrogen peroxide (H 2 O 2 ); lipid peroxyl radical (ROO·); lipid alkoxyl radical (RO); lipid hydroperoxide (ROOH); nitrogen di-oxide (·NO 2 ); nitric oxide (•NO); peroxynitrite (ONOO-) and thiol radical (RS) [32,33].
These free radicals generate oxidative stress and damage proteins, lipids and nucleic acids. Therefore, the role of free radicals is postulated in different disease conditions, such as the process of aging, various cancers, inflammatory conditions (adult respiratory diseases, arthritis and vasculitis), neurological disorders (Alzheimer's disease, Parkinson's disease) and ischemic diseases (stroke, heart diseases and intestinal ischemia) [34].
The antioxidant activity of E. sativa crude extract was evaluated using very wellestablished techniques, viz., DPPH and H 2 O 2 assay. The results of the antioxidant assay reveal that E. sativa crude extract can scavenge the radicals to a great extent. The scavenging efficacy of crude extract on DPPH (IC 50 = 66.16 µg/mL) radicals was better when compared to H 2 O 2 (IC 50 = 76.05 µg/mL). Furthermore, the antioxidant potential of E. sativa crude extract is dose dependent (Figure 4).
Antioxidants are substances that protect the cells from free radicals. Inside the cell, antioxidant molecules are present at low concentrations, which considerably reduces the oxidative stress and prevents the cells from the damage of free radicals [35]. Apart from endogenous antioxidants, they can also be available exogenously from the diet or as dietary supplements. An efficient antioxidant can readily absorb and remove free radicals and chelate metals at a physiologically appropriate level. Usually, endogenous antioxidants maintain optimal cellular functions, but under the condition of oxidative stress, endogenous antioxidants are not enough and exogenous antioxidants are needed to maintain optimal cellular functions [36]. Plants are considered as the potent source of antioxidant molecules, as they synthesize and accumulate various non-enzymatic antioxidants, such as ascorbic acid, glutathione and phenolics and flavonoids. accumulated in the cells and peripheral blood via acting as a precursor of sulforaphane, due to the presence of phenolics and glucosinolates in high concentrations in the seed [38]. molecules. The activity was carried out in triplicate and the data represent the mean ± SD.

The Anticancer Activity of E. sativa Crude Extract
The anticancer potential of E. sativa crude extract was evaluated against two different human colorectal cancer cells (HCT-116 and Caco-2) via MTT assay. The results of the anticancer activity reveal that E. sativa crude extract could significantly inhibit the cell viability of both cells in a dose-dependent manner. The viability of the HCT-116 cell line after the treatment of crude extract was found to be higher, and the IC50 value was calculated, i.e., 64.91 μg/mL compared to Caco-2 with the IC50 value of 83.98 μg/mL ( Figure 5).
After cardiovascular diseases, cancer has been reported to be the most common cause of mortality in several countries. In males, lung, liver, colorectal, prostate and stomach cancer are found to be the most common, whereas in females, breast, cervical, lung, The antioxidant potential of E. sativa crude extract was analyzed against DPPH and H 2 O 2 molecules in comparison to ascorbic acid. The crude extract from E. sativa exhibited notable free radical scavenging activity against both DPPH and H 2 O 2 molecules. Previously, the antioxidant activity of E. sativa seeds was tested by Kishore et al. (2016) by using various in vitro antioxidant methods, such as phenolic content, total antioxidant capacity, reducing power, hydrogen peroxide, nitric oxide and superoxide dismutase scavenging activity. According to the previous study, the total phenol content of alcohol extract and hydro-alcohol extract was found to be 216.0 and 229.00 mg/g of gallic acid equivalents, respectively, and the total antioxidant capacity was determined to be 111.00 and 230.60 µM/g of ascorbic acid equivalents. Alcohol extracts and hydro-alcohol extracts were found to be able to scavenge DPPH radicals, hydrogen peroxide radicals, nitric oxide radicals and superoxide radicals, respectively. The IC 50 values were found to be 3.28 and 3.53 µg/mL, 188.11 and 181.56 µg/mL, 73.05 and 64.33 µg/mL, and 87.91 and 41.12 mg/mL, respectively [37]. Koubaa et al. (2015) reported the antioxidant potential of E. sativa leaf extract and concluded that antioxidant activity is present due to the high concentration of phenolics(kaempferol 3,4-di-O-glucoside, kaempferol 3-glucosyl, quercetin 3-glucosyl and isorhamnetin 3-glucosyl) [14]. In one more study, Maia et al. (2015) reported the antioxidant potential of the seed extract of E. sativa by scavenging H 2 O 2 and alkyl hydroperoxides accumulated in the cells and peripheral blood via acting as a precursor of sulforaphane, due to the presence of phenolics and glucosinolates in high concentrations in the seed [38].

The Anticancer Activity of E. sativa Crude Extract
The anticancer potential of E. sativa crude extract was evaluated against two different human colorectal cancer cells (HCT-116 and Caco-2) via MTT assay. The results of the anticancer activity reveal that E. sativa crude extract could significantly inhibit the cell viability of both cells in a dose-dependent manner. The viability of the HCT-116 cell line after the treatment of crude extract was found to be higher, and the IC 50 value was calculated, i.e., 64.91 µg/mL compared to Caco-2 with the IC 50 value of 83.98 µg/mL ( Figure 5). sativa against melanoma cells. The seed oil (isothiocyanates rich) was found to significantly reduce the tumor growth and angiogenesis in mice without any major toxicity [19]. Azarenko et al. (2014) found that erucin prevents the proliferation of MCF7 (breast cancer cells) (IC50 = 28 mM) in parallel with cell cycle arrest at mitosis (IC50 = 13 mM) and apoptosis by a mechanism consistent with the impairment of microtubule dynamics [40]. However, a further exploration using different solvent system extractions and the in vitro anticancer activity of selected cancer cell lines is still required for the therapeutic implications to improve human health.

The Pharmacokinetic and Toxicity (ADMET) Profiles of the Identified Phytoconstituents from E. sativa Ethanolic Crude Extract
The potential of a good drug could be ruined because of the limited absorption, distribution, metabolism, excretion and toxicity (ADMET) characteristics. Furthermore, the major drawback of drug discovery in clinical trials is believed to be its pharmacokinetic properties, by virtue of which it becomes very expensive. Therefore, ADMET parameters were estimated using in silico tools to determine the probability of the E. sativa ethanolic crude extract becoming a potential candidate for the development of drugs (Tables 2 and  3). Interestingly, the majority of the phytoconstituents were found to meet the Lipinski's rule of five, and some of them were also found to follow Ghose, Veber and Egan filters, with most of them attaining a good score of bioavailability. One more important attribute is the solubility for the absorption of the compound and its distribution in the body, which was specified via the value of aqueous solubility. In the results, it can be observed that most of the compounds are highly soluble in water.
It is important to assess the skin's permeability, i.e., the rate of a molecule penetrating After cardiovascular diseases, cancer has been reported to be the most common cause of mortality in several countries. In males, lung, liver, colorectal, prostate and stomach cancer are found to be the most common, whereas in females, breast, cervical, lung, thyroid and colorectal cancer are the most common [39]. Therefore, there is an urgent need to find alternate and cost-effective types of cancer treatments with lesser or no side effects. Natural products came to the attention of scientists due to their ability to serve as therapeutic as well as preventive agents over the past decade. Almost 60% of all conventional drugs used in cancer treatment are directly or indirectly extracted from plants, which encourages the discovery of medicinal plant-based novel drugs [39]. There are literatures which states, that the regular consumption of the cruciferous vegetable E. sativa can reduce the risk of different types of cancer development in the body.
Several phytochemicals, such as glucosinolates, sulfur containing plant secondary metabolites and isothiocyanate, including sulforaphane and erucin, are believed to be present in E. sativa. Erucin [1-isothiocyanato-4-(methylthio)butane], which is metabolically and structurally similar to sulforaphane, which is also reported to be present in large quantities in E. sativa [40]. The promising anticancer effect of erucin was already reported through many in vitro and in vivo studies. In order to observe the protective effect of erucin against cancer, in vivo research was conducted for the first time by a group of researches on the different mouse tissues mediated through the induction of several detoxification enzymes [41]. To date, many different studies confirmed the effectiveness of erucin against several human cancer cells. The anticancer effect of erucin was reported for different human cancer cell lines, such as the lung, liver, colon, and prostate, with the help of different mechanisms, viz., cell cycle regulation, apoptosis and the inhibition of proliferation as well as mitochondrial depolarization.
One of the studies stated that the erucin can induce apoptosis and also cell cycle arrest on human leukemia cells, together with the multidrug-resistant alternatives [19]. Jakubikova et al. (2005) investigated the anticancer property of erucin, which, similar to sulforaphane, induces the phase II detoxification enzymes and inhibits phase I enzymes. In addition, erucin arrests cell cycle progression and induces apoptosis in human lung carcinomas, hepatomas and leukemia cell lines. Erucin increases the expression of multidrug resistance transporters in human carcinoma cell lines [42]. Nazif et al. (2010) evaluated the cytotoxic activity of the total alcoholic extract of the defatted seeds and the isolated compounds against several types of tumor cell lines using the SRB assay. The total alcoholic extract and the aglucones of the isolated compounds glucoerucin and glucoiberin exhibited significant cytotoxic activity for HCT116 (the colon carcinoma cell line) (IC 50 = 0.74, 2.42 and 0.94 µg/mL), respectively, while the IC 50 was >10 µg/mL for Hela (the cervix carcinoma cell line), HEPG2 (the liver carcinoma cell line), MCF7 (the breast carcinoma cell line) and U251 (the brain carcinoma cell line) [7].
Melchini and Maria (2010) revealed that the intake of vegetables is associated with a reduced risk in the development of various types of cancer. This is attributed to the bioactive hydrolysis products that are derived from these vegetables, namely isothiocyanates. Isothiocyanates are characterized as small organic compounds synthesized as glucosinolates with R-N=C=S functional groups. Isothiocyanates present in cruciferous vegetables have a higher amount of anti-cancerous properties and can inhibit cell proliferation. Isothiocyanates suppress cancer cell proliferation by inhibiting the proteins involved in tumor initiation and proliferation pathways. Meanwhile, isothiocyanate treatment stimulates the reactive oxygen species (ROS), cell cycle arrest, programmed cell death and autophagy. More than 20 isothiocyanates are reported as having anticarcinogenic properties against tumorigenesis [43]. Erucin, the isothiocyanate product derived from E. sativa, showed chemoprevention activity against cancer cells in animal models. The mechanism of action showed the modulation of phase I, II and III detoxification, the regulation of cell growth by the induction of apoptosis, cell cycle arrest, the induction of ROS mechanisms and regulation androgen receptor pathways [13]. Michael et al. (2011) reported the potent anticancer activity of a 70% ethanolic extract of E. sativa against different human tumor cell lines, such as HepG2 (liver carcinoma), MCF7 (breast carcinoma), HCT116 (colon carcinoma) and Hep2 (larynx carcinoma) [19]. Khoobchandani et al. (2011) investigated the anticancer potential of solvent extracts prepared from the aerial roots and seed oil of E. sativa against melanoma cells. The seed oil (isothiocyanates rich) was found to significantly reduce the tumor growth and angiogenesis in mice without any major toxicity [19]. Azarenko et al. (2014) found that erucin prevents the proliferation of MCF7 (breast cancer cells) (IC 50 = 28 mM) in parallel with cell cycle arrest at mitosis (IC 50 = 13 mM) and apoptosis by a mechanism consistent with the impairment of microtubule dynamics [40]. However, a further exploration using different solvent system extractions and the in vitro anticancer activity of selected cancer cell lines is still required for the therapeutic implications to improve human health.

The Pharmacokinetic and Toxicity (ADMET) Profiles of the Identified Phytoconstituents from E. sativa Ethanolic Crude Extract
The potential of a good drug could be ruined because of the limited absorption, distribution, metabolism, excretion and toxicity (ADMET) characteristics. Furthermore, the major drawback of drug discovery in clinical trials is believed to be its pharmacokinetic properties, by virtue of which it becomes very expensive. Therefore, ADMET parameters were estimated using in silico tools to determine the probability of the E. sativa ethanolic crude extract becoming a potential candidate for the development of drugs (Tables 2 and 3). Interestingly, the majority of the phytoconstituents were found to meet the Lipinski's rule of five, and some of them were also found to follow Ghose, Veber and Egan filters, with most of them attaining a good score of bioavailability. One more important attribute is the solubility for the absorption of the compound and its distribution in the body, which was specified via the value of aqueous solubility. In the results, it can be observed that most of the compounds are highly soluble in water.
It is important to assess the skin's permeability, i.e., the rate of a molecule penetrating the stratum corneum, to determine the potential for creating a form of transdermal drug delivery. It is considered that a molecule will penetrate the skin at the log Kp value of more than −2.5 cm/h. All of the identified phytochemical constituents from the crude extract were found to possess moderate-to-good skin penetrability. Caco-2 is the human epithelial colorectal adenocarcinoma cell and its permeability can calculate the intake of oral drugs. Most of the compounds were found to have moderate-to-potent Caco-2 permeability values (log Papp values > 0.90 cm/s).
ADMET analysis is a computer-based drug designing approach, which can lead to the initial stage of drug discovery [44][45][46]. The main motive behind this in silico approach is to lower the cost and time factors involved in comparison to standard ADMET profiling, as the in silico approach can screen more than 20,000 compounds within a minute; the wet lab will take more than 20 weeks to perform the same task [47][48][49]. Therefore, recently, after the establishment of ADMET data in the 1990s, the majority of pharmaceutical companies used this computational approach for the screening of drugs. In the present study, we also performed a computer-aided prediction of pharmacokinetic properties and the safety profile of the identified phytoconstituents via SwissADME and pkCSM tools. The results of the computational analysis can be useful for researchers to advance the development of prospective semi-synthetic and synthetic drugs for miscellaneous use.
Most of the identified phytochemical compounds were not found as a P-gp inhibitor/substrate. P-gp is the main element of ATP-binding cassette transporters or ABCtransporters, which is utilized to protect the central nervous system (CNS) from xenobiotics and is a prime method used to determine active efflux through biological membranes. The majority of the phytochemical compounds were found to be absorbed by the intestine during intestinal absorption analysis. The prediction of skin permeation was carried out by LogKP, by which phytochemical constituents were identified and found to penetrate the skin at a moderate or high level, which confirms their drug-like feature. Compounds 1, 3, 5 and 16 have a log BB (logarithm value of the brain-to-plasma concentration ratio, 0.1 to 0.3) and were determined to have more potential to cross the brain blood barrier (BBB). Only fewer compounds have the ability to penetrate the CNS. The compounds 9, 28, 31 and 43 are amongst the phytoconstituents that are more successfully distributed, with a distribution volume (logVDss) of 0.562 L/kg and 1.444 L/kg, respectively, in the tissues.    Table 3. Absorption, distribution, metabolism, excretion and toxicity (ADMET) properties of the identified phytochemical compounds of E. sativa:    The isoforms of human cytochrome P450 (CYP), which are integrated in the metabolism of drugs inside the liver were also assessed. The foremost clinically appropriate drugmetabolizing enzyme in the human body is CYP3A4. The inhibition of CYP3A4 could direct lead to drug toxicity, drug-drug interactions and other adverse effects. Some of them were non-inhibitors/substrates of isoenzymes. Most of the compounds were found to be non-inhibitors of CYP3A4, the isoenzyme responsible for the metabolism of about 60% of xenobiotics, including drugs, carcinogens, steroids and eicosanoids.
To predict the route of excretion, the total clearance (CLTOT) for both hepatic and renal and renal organic cation transporter 2 (OCT2) substrates was expressed as the log mL/min/kg that was predicted. The results revealed that the majority of the phytochemical constituents displayed a positive total clearance value and can be simply be excreted. The AMES toxicity, hepatotoxicity, hERG potassium channel inhibition and skin sensitization parameters were predicted to find out the toxicity profile of the identified phytochemical constituents from the E. sativa crude extract. The obtained results reveal that only a few of the compounds have a deviated mutagenic and hepatic toxicity potential, which means that the majority of the compounds are devoid of any risk of toxicity (99% have no hERG I inhibition and 72% exert no skin-sensitive effects).
To obtain further information about the improved bioavailability and drug-likeness of the identified phytochemicals, we estimated the results of the bioavailability radar. The results are represented by the lipophilicity: XLOGP3 between −0.7 to +5.0; polarity: TPSA between 20-130 Å 2 ; size: molecular weight 150-500 g/mol; saturation: fraction of carbons in the sp3 hybridization not less than 0.25; solubility: log S not higher than 6; and flexibility: no more than 9 rotatable bonds with the colored zone representing the desired physicochemical space for good oral bioavailability, and they all display significant drug-likeness properties (Figures 6 and 7).
The examples of the brain or intestinal estimated permeation (BOILED Egg model) estimation of gastrointestinal (GI) absorption and BBB permeation for all the identified compounds were conducted. The results show that the compound indicated with a red point in the yellow ellipse has the potential for brain penetration and is a non-substrate of P-gp (PGP-) ( Figure 8).
Some of the plant families, such as Brassicaceae, Capparaceae and Resedaceae, were reported to contain glucosinolates, which are considered as one of the most significant secondary metabolites. Subsequently, in addition to the enzyme myrosinase, secondary metabolites are broken down into various hydrolytic products [50,51]. E. sativa, a member of the Brassicaceae family, is reported to be a rich source of glucosinolates, and its different hydrolytic products are reported to exert different bioactive properties, such as anticancer, antimutagenic, bioherbicidal, antimicrobial, antigenotoxic and antitumor activities [52][53][54][55][56]. In the present study, two glucosinolates, glucoraphanin and 4-dimethoxyglucobrassicin, were identified, which could be the major constituents of the E. sativa crude extract and are possibly responsible for various potent biological activities.
The examples of the brain or intestinal estimated permeation (BOILED Egg model) estimation of gastrointestinal (GI) absorption and BBB permeation for all the identified compounds were conducted. The results show that the compound indicated with a red point in the yellow ellipse has the potential for brain penetration and is a non-substrate of P-gp (PGP-) (Figure 8).    Some of the plant families, such as Brassicaceae, Capparaceae and Resedaceae, were reported to contain glucosinolates, which are considered as one of the most significant secondary metabolites. Subsequently, in addition to the enzyme myrosinase, secondary metabolites are broken down into various hydrolytic products [50,51]. E. sativa, a member of the Brassicaceae family, is reported to be a rich source of glucosinolates, and its different hydrolytic products are reported to exert different bioactive properties, such as anticancer, antimutagenic, bioherbicidal, antimicrobial, antigenotoxic and antitumor activities [52][53][54][55][56]. In the present study, two glucosinolates, glucoraphanin and 4-dimethoxyglucobrassicin, were identified, which could be the major constituents of the E. sativa crude extract and are possibly responsible for various potent biological activities. Figure 8. BOILED Egg model of the phytochemical compounds of E. sativa using the Swiss ADME predictor. BOILED Egg allows for the intuitive evaluation of passive gastrointestinal absorption (HIA) and brain penetration (BBB) in function of the position of the molecules in the WLOGPversus-TPSA referential. The white region indicates the high probability of passive absorption by the gastrointestinal tract, and the yellow region (yolk) indicates the high probability of brain penetration. The yolk and white areas are not mutually exclusive. In addition, the points are colored in blue if predicted as actively effluxed by P-gp (PGP+) and in red if predicted as non-substrate of P-gp (PGP-).

Media and Chemicals
All the microbiological media used in the present study were purchased from the local suppliers of Hi-Media ® , Mumbai, India. Chemicals, drugs and solvents were purchased from Sigma-Aldrich ® , Bangalore, India. Analytical grade solvents were used.

Plant Material and Extraction Preparation
E. sativa plants were cultivated in a field for the purpose of the present study. Firstly, the plants of E. sativa were grown in silty clay with sand soil type at an average temperature of 30-35 • C, and were watered once every day. Later, the whole plant leaves were collected after maturity, washed under running tap water and oven-dried. Dry plant leaves were then grounded into a fine powder with an electrical grinder and stored in airtight containers. Next, E. sativa powder (25 g) was soaked in 85% ethanol at 37 • C for 24 h with vigorous shaking at 120 rpm. The ethanolic extract was filtered using Whatman no. 1 filter paper and the obtained extract was concentrated using a rotary evaporator. The obtained dried residues were dissolved in 10% dimethyl sulfoxide (DMSO) to make up 1 mg/mL of plant leaf extract concentration to carry out various biological assays [57].

High Resolution Liquid Chromatograph Mass Spectrometery Analysis
The HR-LCMS analysis of the ethanolic extract was carried out by a UHPLC-PDA-Detector Mass Spectrophotometer (HR-LC/MS 1290 Infinity UHPLC System), Agilent Technologies ® , Santa Clara, CA, USA, and consisted of an HiP sampler, binary gradient solvent pump, column compartment and Quadrupole Time of Flight Mass Spectrometer (MS Q-TOF) with a dual Agilent Jet Stream Electrospray (AJS ES) ion source. A total of 1% formic acid in deionized water (solvent A) and acetonitrile (solvent B) was used as a solvent. The flow rate of 0.350 mL/min was used, while MS detection was performed in MS Q-TOF. Compounds were identified via their mass spectra and their unique mass fragmentation patterns. Compound Discoverer 2.1, ChemSpider and PubChem were used as the main tools for the identification of the phytochemical constituents [58].

Antibacterial Assay
All pathogenic bacterial test strains, Pseudomonas aeruginosa (P. aeruginosa) (MTCC 741), Bacillus subtilis (B. subtilis) (MTCC 121), Escherichia coli, (E. coli) (MTCC 9537) and Staphylococcus aureus (S. aureus) (MTCC 96), were obtained from the Microbial Type Culture Collection (MTCC), India, and Muller-Hinton Agar (MHA) was used to maintain the bacterial culture. The antibacterial activity of E. sativa crude extract was carried out via the agar cup/well diffusion method. Firstly, bacterial cultures were grown overnight at 37 • C in fresh medium and a total of 0.5 McFarland standard 10 8 colony-forming units/mL (CFU/mL) was matched by culture turbidity adjustment using 0.9% of sterile saline solution. Bacterial suspension was evenly spread all over the plates, and the wells were made with a sterile cork borer. A total of 60 µL of crude extract (1 mg/mL) was then inoculated into each respective well and the plates were incubated at 37 • C for 24 h. Antibacterial activity was noted in the form of the zone of inhibition. A total of 1000 µg/mL of chloramphenicol and 10% of DMSO was used as a positive and negative control, respectively [58].

DPPH Scavenging Activity
The antioxidant potential of E. sativa crude extract was determined in terms of its radical scavenging capability against DPPH free radicals [59]. Crude extracts of different concentrations (1-100 µg/mL) were mixed in the tubes containing 2 mL of DPPH solution (6 × 10 −5 M) in DMSO. The tubes were mixed well and incubated in the dark for 1 h. At the end of the incubation, the absorbance was read out at 517 nm. DMSO was used as a blank, DPPH solution without any crude extract was used as a control, and ascorbic acid was used as a standard [60]. The calculation of the percentage of scavenging of DPPH free radicals was calculated as follows: DPPH scavenging activity (%) = (A0 − A1)/A0 × 100 (1) where A0 = absorbance of the control; A1 = absorbance of the sample.

Hydrogen Peroxide Scavenging Activity
The antioxidant potential of E. sativa crude extract against H 2 O 2 was carried out according to the described method [59]. Crude extract of different concentrations (1-100 µg/mL) were mixed in the tubes having 1 mL of H 2 O 2 (1 mL, 2 mM) solution, prepared in a phosphate buffer (0.1M, pH 7.4). The tubes were then incubated for 10 min at room temperature. At the end of the incubation, the absorbance was read out at 230 nm against a blank solution (phosphate buffer without H 2 O 2 ), while ascorbic acid was used as a positive control [61]. The following formula was then used to calculate the percentage of H 2 O 2 scavenged: Inhibition (%) = (A0 − A1)/A0) × 100 (2) where A0 = absorbance of the control; A1 = absorbance of the extract/standard

Anticancer Assay (MTT Assay)
The anticancer activity of E. sativa crude extract was carried out against human colon cancer cell lines (HCT-116 and Caco-2). Both cells were acquired from the National Centre for Cell Science (NCCS), India, and propagated in 25 cm 2 flask containing Dulbecco's Modified Eagle's Medium (DMEM) supplemented with 10% Fetal Bovine Serum (FBS) and antibiotic solution in a humidified (5% CO 2 ) atmosphere at 37 • C. To perform the MTT assay, cells were grown up to 80% confluence and then seeded at a density of more than 1 × 105 cells per well in 96-well plates and incubated in conditions, as mentioned above. Trypan Blue (0.4%) was used to stain the cells, and the viability was calculated by using a hemocytometer. Cells were then treated with different concentrations of E. sativa crude extract (1-100 µg/mL) for 24 h. Cells were washed with PBS solution and subjected to 100 µL of MTT solution (3-(4,5-dimethylthiazolyl-2)-2,5 diphenyltetrazolium bromide) (5 mg/mL) and incubated for 4 h. Then, the medium was removed and 100 µL of DMSO was added to solubilize the formazan crystals. The amount of formazan crystal was determined by measuring the absorbance at 570 nm using an ELISA reader. Cisplatin was used as a positive control. All assays were performed in triplicate and 50% cytotoxic concentration (IC 50 ) was calculated [62,63].

Conclusions
From the present study, it can be concluded that E. sativa displays potent antibacterial activity against the different human pathogenic bacterial strains. Additionally, E. sativa also shows promising antioxidant and anticancer activity. Based on our results, E. sativa can also be utilized for the production and development of nutraceutical or functional food. Therefore, further investigation warrants in vivo pharmacological and toxicological research to demonstrate the unexplored and valuable aspect of E. sativa. The computational analysis shows that E. sativa possesses a significant pharmacokinetic and safety profile of the phytochemical constituents identified using HR-LC/MS. Additionally, it also reveals the ethanolic crude extract of E. sativa as a prospective drug candidate for the treatment and management of various diseases, as well as for therapeutic purposes. However, the current computational models are severely constrained due to the lack of understanding of the underlying molecular processes of the diseases. Therefore, to overcome these limitations, one way to formulate more effective strategies is to incorporate ligand, target, phenotype and biological network-based approaches, as well as a deeper reinforcement of learning methods, which are more likely to increase predictability. This will overcome the shortcomings of the existing computational approaches and improve the drug development process.  Data Availability Statement: All data generated or analyzed during this study are included in this article.